[CANCER RESEARCH 41, 2991-2994, August 1981] 0008-5472/81 /0041-OOOOS02.00 Inhibition of Carcinogen-induced Neoplasia by Sodium Cyanate, ferf-Butyl Isocyanate, and Benzyl Isothiocyanate Administered Subsequent to Carcinogen Exposure1
Lee W. Wattenberg
Department of Laboratory Medicine and Pathology. University of Minnesota, Minneapolis, Minnesota 55455
ABSTRACT of polycyclic aromatic hydrocarbons inhibits the occurrence of neoplasia (21). The present investigation was directed towards The effects of sodium cyanate, ferf-butyl isocyanate, and determining if sodium cyanate would be inhibitory if its feeding benzyl isothiocyanate on carcinogen-induced neoplasia were were started shortly after carcinogen administration, the period studied in experiments in which the test compound was fed during which neoplasia evolves. Two animal systems have been starting 1 week following completion of carcinogen administra used, i.e., DMBA2-induced mammary neoplasia in the rat and tions. Under these conditions, all three test compounds exerted DMH-induced neoplasia of the large intestine in the mouse. In an inhibitory effect on the occurrence of 7,12-dimethyl- both, sodium cyanate was fed in the diet starting 1 week after benz(a)anthracene-induced neoplasia of the breast of carcinogen exposure and continued throughout the subse Sprague-Dawley rats. In a second experiment model, sodium quent course of the experiment. In addition to sodium cyanate, cyanate inhibited the occurrence of 1,2-dimethylhydrazine-in- 2 other compounds, fe/t-butyl isocyanate and benzyl isothio duced neoplasia of the large bowel of female CF-1 mice. Thus, cyanate, were investigated. a new group of compounds has been identified which has inhibitory capacities against neoplasia when given subsequent MATERIALS AND METHODS to carcinogen exposure. Mammary Neoplasms. The procedure for producing mam INTRODUCTION mary tumors was a modification of that described by Muggins et al. (8). Female Sprague-Dawley rats (King Company, Ore An increasing number of compounds are being found to gon, Wis., or Holtzman Company, Madison, Wis.) were given inhibit chemical carcinogenesis (14, 15, 18-22). One of these 12 mg of DMBA in 1.0 ml of olive oil by p.o. intubation when inhibitors has been identified recently as sodium cyanate (21). they were 7 weeks old. One week later, the rats were random This compound is of particular interest because it produces a ized by weight and placed on the experimental diets. These marked inhibition of protein synthesis in a variety of neoplastic diets consisted of powdered Purina rat chow (Ralston Purina cells under conditions where a corresponding inhibition of Company, St. Louis, Mo.) containing the test compound or protein synthesis in normal tissues of the tumor-bearing animal without any additions for the diet fed the control groups. The does not occur. Neoplastic cells in which inhibition of protein experimental diets were fed until the experiment was termi synthesis has been observed include hepatomas, MK-3 kidney nated. The rats were weighed at 2-week intervals, and mam tumors, primary and transplantable carcinomas of the large mary tumors were counted starting 10 weeks after administra bowel of the rat, and Ehrlich ascites cells (1, 4, 5, 12, 13). tion of DMBA. The diagnosis was confirmed at autopsy. Sodium cyanate requires metabolic activation in order to inhibit Neoplasms of the Large Bowel. The procedure for produc protein synthesis. A phenobarbital-inducible cytochrome P- ing large-bowel neoplasms was a modification of that described 450 has been implicated as the activating system (5). Data by Thurnherr ef al. (16). Female CF-1 mice (Charles River have been obtained indicating that suppression of protein Breeding Laboratory, Kingston, N. Y.) were used throughout. synthesis is the result of inhibition of protein chain initiation (1 ). When the mice were 10 weeks old, they were randomized by In addition to the effects on protein synthesis, sodium cyanate weight. At that time, 0.6 mg of DMH in a 0.2-ml solution of has also been shown to inhibit DNA synthesis in neoplastic EDTA brought to pH 6.5 with sodium carbonate was injected cells (10-12). Another property of sodium cyanate is that it s.c. A total of 16 DMH administrations, twice a week for 8 carbamylates the amino-terminal residues of both the a- and weeks, was given. One week following the last injection of /?-globin chains of hemoglobin S (7). The compound has been DMH, the mice were placed on the experimental diets. These found to have anti-sickling properties and has been proposed diets consisted of powdered Purina rat chow containing the as a potential treatment for sickle cell disease. Although test compound or without any additions for the diet fed the chronic administration studies in animals have shown low tox- control groups. The experimental diets were fed until mice icity, some adverse effects in humans have limited its useful were 52 weeks old. At that time, all animals were killed by ness (2, 3, 6, 7). inhalation of CO2 and autopsied. The large bowel was opened, Previous work has shown that administration of sodium cy and the number and location of neoplasms were recorded. anate in the diet prior to and during the course of administration Each lesion was studied microscopically. Chemicals. Sodium cyanate was obtained from the Aldrich 1 Supported by USPHS Research Grants CA-15638 through the National Large Bowel Cancer Project and CA-14146, both from the National Cancer Institute. 2 The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene; DMH, Received February 13, 1981; accepted April 22, 1981. symmetrical 1,2-dimethylhydrazine dihydrochloride.
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Chemical Company, Milwaukee, Wis. The preparation was with fert-butyl isocyanate and benzyl isothiocyanate as the test assayed as follows: sodium cyanate, 97%; sodium chloride, compounds. 3%; and sodium carbonate, trace. Other chemicals used were The experiments determining the inhibitory capacity of so as follows: DMH and ferf-butyl isocyanate (Aldrich); and benzyl dium cyanate on DMH-induced neoplasia of the large bowel isothiocyanate and DMBA (Eastman Organic Chemicals, Roch showed that feeding sodium cyanate resulted in fewer mice ester, N. Y.). having demonstrable neoplasms at the conclusion of the ex Statistical Analysis. Student's f test was used to determine periments and a decrease in the average number of neoplasms the statistical differences in the number of tumors per group per mouse. The magnitude of the protective effect was similar between the control and treated groups, and the x2 test was to that found in the mammary tumor protocols. If the results of used for the differences of percentage of tumor-bearing ani Experiments 1 and 2 are combined, 39% as many mice fed mals in these groups. sodium cyanate (0.026 mmol/g of diet) had neoplasms of the large bowel as compared to the control groups. The average RESULTS number of neoplasms in the sodium cyanate groups was 38% that of the control (Table 2). Data on the effects of sodium cyanate on DMBA-induced mammary neoplasia in the rat are shown in Table 1. In all 3 DISCUSSION experiments in which it was used, sodium cyanate administra tion resulted in fewer rats showing mammary neoplasms and a Sodium cyanate has been shown to inhibit protein and DNA decrease in average number of tumors per rat. Comparable synthesis in several types of neoplastic cells, including those findings were obtained with rats fed ferf-butyl isocyanate and of the large bowel. It is possible that the mechanism by which benzyl isothiocyanate. The 4 experiments were carried out the early stages of neoplasia of the large bowel and mammary over a period of 2 years. As described in "Materials and gland were inhibited in the present study resides in the capacity Methods," Sprague-Dawley rats from 2 different suppliers were of sodium cyanate to suppress synthesis of one or both of used. Those used in Experiments 3 and 4 were more respon these macromolecular species. The cyanate anión undergoes sive to DMBA. With the mammary tumor count at a shorter time activation to an unstable metabolite by a phenobarbital-induc- interval, the data become comparable to those in Experiments ible cytochrome P-450 system (5). Thus, the magnitude of the 1 and 2. When all the rats in the control groups in Experiments inhibitory activities observed in the present study might be 1 to 3 showed mammary tumors (with the exception of one controlled by the activity of the activating system. If this is the animal in Experiment 1), neoplasms were found in 60 to 63% case, the potential would exist for magnifying the inhibitory of rats in the groups fed 0.026 mmol of sodium cyanate per g effects of sodium cyanate by enhancing the activity of the of diet. The average number of tumors per animal in the sodium activating system. cyanate groups was 42 to 43% of that of the corresponding The finding that ferf-butyl isocyanate and benzyl isothiocya controls. Thus, in the 3 experiments, consistent results were nate cause levels of inhibition of mammary tumor formation obtained. In Experiment 4, similar inhibitory effects were found comparable to those produced by sodium cyanate suggests
Table1 Effects of sodium cyanate and related compounds on DMBA-induced mammary tumor formation in female Sprague-Dawley rats neoplasms0No.
Experi tration of gain
±0.4 SodiumcyanateNone 1629 6666 639100 1.0 ±0.3"2.8
±0.2 Sodium cyanate 13 646553 779 1.2 ±0.3 60"10050"63"1 1 .2 ±0.5"3.4 SodiumcyanateNone 0.0260.013 1527
±0.3 1.2 ±0.4h rert-Butyl ¡socyanate 16 43 0.4hCompany,1.4 ± ¡sothiocyanateaBenzyl 16used0.017Mammary 48% ratsExperimerConcenFemale Sprague-Dawley Experimentsits in Kingexperiments,DMBA,and 2 were from inig those 3 and 4 were from Holtzman Compamy. In allWt 12 rrNo.in 1.0 ml olive oil, was given by P.O. intubation when the rats were 7 weeks old. The experimental diets were begun at 8 weeks of age and continued throughout the remainder of the experiment. 6 Weight gain from 7 to 25 weeks of age in Experiments 1 and 2 and from 7 to 23 weeks of age in Experiments 3 and 4. 0 Mammary neoplasms when rats were 25 weeks old in Experiments 1 and 2 and 23 weeks old in Experiments 3 and 4. d Number of neoplasms occurring in the entire group divided by the number of rats at risk. 8 Mean ±S.E. 'p<0.05. 9p<0.01. " p < 0.001.
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Table 2 Effects of sodium cyanate on DMH-induced neoplasia of the large bowel
Neoplasms of the large bowel
All neoplasms Adenocarcinomas Adenomas
tration of gain with of tumors/ mice with of tumors/ mice with of tumors/ Experiment312Combined
±0.200.22 ±0.080.06 ±0.170.17 cyanateNoneSodium ±0.130.94 ±0.050.19 ±0.120.75
1and ±0.150.36 ±0.050.06 ±0.140.31 2Additions cyanateConcen ±0.16%of ±0.04%of ±0.149 Female CF-1 mice were given 16 s.c. administrations of 0.6 mg DMH (twice a week for 8 weeks) starting when the animals were 10 weeks old. One week after the last administration of DMH, the mice were placed on the experimental diets, and they were continued on these diets until the animals were 52 weeks old. At that time, the experiments were terminated. 6 Weight gain from 10 to 52 weeks of age. c Neoplasms of the large bowel when mice were 52 weeks old. d Adenocarcinomas plus adenomas of the large bowel. " Number of neoplasms in the entire group divided by the number of mice at risk. 'Mean ±S.E. 9 p < 0.05. "p<0.01.
that the suppression of early neoplastia events may be brought 2692-2698, 1977. 2. Alter, B. P., Kan, Y. W., and Nathan, D. G. Inhibition of hemoglobin synthesis about by a diverse group of related compounds. It is not known by cyanate in vitro. Blood, 43: 57-68, 1974. whether the mechanism of inhibition by all 3 compounds is the 3. Birch, K. M., and Schutz, F. Actions of cyanate. Br. J. Pharmacol., 1: 186- same or if they differ. The reactive species of both ferf-butyl 193, 1946. isocyanate and benzyl isothiocyanate could be the undisso- 4. Boffa, L. C., and Allfrey, V. G. Selective inhibition by sodium cyanate of protein synthesis in tumor cells (abstract). J. Cell Biol., 79: 362, 1978. ciated molecules. However, both ferf-butyl isocyanate and 5. Boffa, L. C., Kozak, S., and Allfrey, V. G. Activation of sodium cyanate for benzyl isothiocyanate can undergo dissociation to form the selective inhibition of protein synthesis in cultured tumor cells. Cancer Res., stable ferf-butyl and benzyl carbonium ions. The stability of 41: 60-66, 1981. 6. Cerami, A., Allan, T. A., Graziano, J. H., DeFuria, J. M., Manning, J. M., and these ions is a good driving force for the liberation of the Gillette, P. N. Pharmacology of cyanate, I. General effects on experimental isocyanate and isothiocyanate anions. Once these anions are animals. J. Pharmacol. Exp. Ther., 185: 653-666, 1973. 7. Gillette, P. N., Peterson, C. M., Lu, Y. S., and Cerami, A. Sodium cyanate as formed, their reactivities would be the same as the correspond a potential treatment for sickle-cell disease. N. Engl. J. Med., 290. 654- ing ions from the sodium salt. The isocyanate and cyanate ions 660, 1974. are identical because of resonance. The isothiocyanate ion 8. Muggins, C., Lorraine, G., and Fukunishi, R. Aromatic influences on the yields of mammary cancers following administration of 7,12- would be expected to have reactivities similar to those of the dimethylbenz(a)anthracene. Proc. Nati. Acad. Sei. U. S. A., 51: 737-742, cyanate ion. Studies of Lea and Koch (10) have shown that 1964. sodium thiocyanate resembles sodium cyanate in its inhibitory 9. Kjaer, A. Naturally occurring ¡sothiocyanates and their parent glycosides. In: N. Kharasch (ed.), Chemistry of Organic Sulfur Compounds, Vol. 1, pp. effects on thymidine incorporation and on the uptake of phos 409-420. Elmsford, N. Y.: Pergamon Press, 1961. phate and amino acids in transplanted tumors of the rat. 10. Lea, M. A., and Koch, M. R. Effects of cyanate, thiocyanate and amygdalin on metabolite uptake in normal and neoplastic tissues of the rat. J. Nati. Accordingly, it is possible that the 3 compounds found to inhibit Cancer. Inst.. 63. 1279-1283, 1979. neoplasia in the present work act through a common mecha 11. Lea, M. A., Koch, M. R., Allfrey, V. G., and Morris, H. P. Inhibition by cyanate nism. of DNA synthesis in hepatomas. Cancer Biochem. Biophys., 1: 129-133, 1975. The full variety and interrelationships of cyanates, isocya- 12. Lea, M. A., Koch, M. R., Beres, B., and Dayal, V. Divergent effects of nates, isothiocyanates, and possibly thiocyanates that will sup cyanate on amino acid and phosphate uptake by liver and hepatoma. Biochim. Biophys. Acta, 474. 321-328, 1977. press the occurrence of neoplasia warrant further exploration. 13. Lea, M. A., Koch, M. R., and Morris, H. P. Tumor-selective inhibition of the Benzyl isothiocyanate is of interest in that it is a naturally incorporation of 3H-labeled amino acids into protein by cyanate. Cancer occurring compound. Benzyl isothiocyanate and the closely Res., 35. 2321-2326, 1975. 14. Slaga, T. J., Klein-Szanto, J. J. P., Fischer, S. M., Weeks, C. E., Nelson, K., related compound, phenethyl isothiocyanate, occur in relatively •and Major, S. Studies on mechanisms of action of anti-tumor-promoting large amounts in cruciferous vegetables including cabbage, agents: their specificity in two-stage promotion. Proc. Nati. Acad. Sci. Brussels sprouts, cauliflower, and broccoli (9, 17). Thus, there U. S. A., 77: 2251-2254. 1980. 15. Sporn, M. B., and Newton, D. L. Chemoprevention of cancer with retinoids. is consumption of at least one category of food containing an Fed. Proc., 28. 84-90, 1979. inhibitor(s) of neoplastic events subsequent to carcinogen ex 16. Thurnherr, N., Deschner, E. E., Stonehill, E. H., and Lipkin, M. Induction of adenocarcinoma of the colon in mice by weekly injections of 1,2-dimethyl- posure. hydrazine. Cancer Res., 33: 940-945, 1973. 17. Virtanen, A. Some organic sulfur compounds in vegetables and fodder plants and their significance in human nutrition. Angew Chem. (Engl.), ): 299-306, REFERENCES 1962. 18. Wattenberg, L. W. Inhibition of carcinogenic effects of polycyclic hydrocar 1. Allfrey, V. G., Boffa, L. C., and Vidali, G. Selective inhibition with sodium bons by benzyl isothiocyanate and related compounds. J. Nati. Cancer Inst., cyanate of protein synthesis in colon cancer cells. Cancer (Prilla.), 40: 58. 395-398, 1977.
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19. Wattenberg, L. W. Inhibitors of chemical carcinogenesis. Adv. Cancer Res., 21. Wattenberg, L. W. Inhibition of polycyclic aromatic hydrocarbon-induced 26. 197-223. 1978. neoplasia by sodium cyanate. Cancer Res., 40: 232-234, 1980. 20 Wattenberg, L. W. Naturally occurring inhibitors of chemical carcinogenesis. 22. Wattenberg, L. W. Inhibitors of chemical carcinogens. In: J. H. Burchenal In: E. C. Miller (ed.), Naturally Occurring Carcinogens—Mutagens and (ed.). Cancer 1980: Achievements. Challenges and Prospects, (pp 517- Modulators of Carcinogenesis. pp. 315-329. Baltimore: University Park 539). New York: GruñeandStratton, 1981. Press. 1979.
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Lee W. Wattenberg
Cancer Res 1981;41:2991-2994.
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